Fmoc-aza-β3-Lys-OAllyl and Fmoc-aza-β3-Asp- OAllyl for on-resin head-to-tail cyclization of aza-β3-peptides

Article abstract of DOI:10.1016/j.tetlet.2012.11.113

A rapid and efficient Fmoc/t-Bu solid-phase peptide synthesis (SPPS) of cyclic aza-β³-peptide analogs by allyl strategy is described. Our synthetic approach includes the synthesis of two new aza-β³- amino acids, Fmoc-aza-β³-Lys-OAllyl and Fmoc-aza- β³-Asp-OAllyl, then the resin attachment of the first aza-β³-amino acid via its side chain, successful use of combination of three orthogonal removable protecting groups, stepwise solid-phase synthesis of linear peptide analog, and on-resin head-to-tail cyclization.

Full text of DOI:10.1016/j.tetlet.2012.11.113

Tetrahedron Letters 54 (2013) 775–778
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Fmoc-aza-b3-Lys-OAllyl and Fmoc-aza-b3-Asp-OAllyl for on-resin
head-to-tail cyclization of aza-b3-peptides
⇑
Shoukri Abbour, Michèle Baudy-Floc’h
ICMV, ISCR, UMR 6226 CNRS, Université de Rennes I, 263 Av. du Général Leclerc, F-35042 Rennes Cedex, France
a r t i c l e i n f o
a b s t r a c t
A rapid and efficient Fmoc/t-Bu solid-phase peptide synthesis (SPPS) of cyclic aza-b3-peptide analogs by
allyl strategy is described. Our synthetic approach includes the synthesis of two new aza-b3-amino acids,
Fmoc-aza-b3-Lys-OAllyl and Fmoc-aza-b3-Asp-OAllyl, then the resin attachment of the first aza-b3-amino
acid via its side chain, successful use of combination of three orthogonal removable protecting groups,
stepwise solid-phase synthesis of linear peptide analog, and on-resin head-to-tail cyclization.
Ó 2012 Elsevier Ltd. All rights reserved.
Article history:
Received 14 September 2012
Revised 8 November 2012
Accepted 12 November 2012
Available online 2 December 2012
Keywords:
Aza-b3-amino acids
Allyl protection
Lysine analog
Aspartique analog
Cyclic pseudopeptides
Solid-phase peptide synthesis (SPPS) is one of the most promis-
ing methods in automated synthesis, which allows rapid access to
peptides. However, peptides are not ideal candidates for many ave-
nues of pharmaceutical development. The main reason peptides
were not used more extensively as pharmaceuticals is that their
bioavailabilities are not high, mainly due to proteolytic degrada-
tion. Due to their enhanced metabolic stability, bioavailability,
and biological absorption, peptidomimetics have been the focus
of research interests over the past several years.1–7
side reactions, and easy purification. In Fmoc/t-Bu strategy, the
allyl protecting group is used as another orthogonal protection
strategy.16–26 The allyl group is generally removed by treatment
of the protected resin with tetrakis (triphenyl-phosphine) palla-
dium (0) [Pd(PPh3)4] in different mixtures of solvents and in the
presence of nucleophiles such as morpholine, N-methylaniline, or
phenyl silane (PhSiH3) the role of which is to scavenge the allyl
group released during the deprotection.27–30
As part of our research program aimed at developing new pep-
tide analogs with potentially useful biological properties, we have
developed a synthesis strategy for aza-b3-peptides.31–36 Here we
described our study on developing a rapid and efficient on-resin
synthesis of cyclic aza-b3-peptides fully compatible with standard
Fmoc/t-Bu chemistry in order to establish a general synthetic route
to these analogs.
Cyclic aza-b3-peptides have been shown as lead compounds for
the development of new antimicrobial agents against fungi, bacte-
ria.8–10 Therefore total SPPS of these important products represents
an important step toward complete exploitation of their therapeu-
tic potential. Poorly chosen places for ring closure can lead to slow
cyclization rates, thus facilitating side reactions such as dimeriza-
tion, oligomerization, and/or epimerization of the C-terminal resi-
due. Traditional method to prepare cyclic peptides11–14 and
therefore cyclic aza-b3-peptides involves SPPS of the selectively
protected linear precursor and cyclization in solution under high
dilution conditions.8–10,15 As an attractive alternative, cyclization
could be performed while peptides still remain anchored to the re-
sin, the on-resin method has been more efficient to prepare cyclic
peptides than the solution-phase approach. The principle of on-re-
sin cyclization is based on anchoring the peptide to the resin
through a side chain and applying orthogonal protecting groups
for ring closure. It possesses advantages, such as time saving, less
Fmoc-aza-b3Lys-OAllyl 4 (R0 = (CH2)4NH2) and Fmoc-aza-b3Asp-
OAllyl 4 (R0 = CH2CO2H) may be useful building blocks for the syn-
thesis of cyclic aza-b3-peptides involving side-chain attachment
and on-resin head-to-tail cyclization (Scheme 1).
N-substituted Fmoc hydrazine
1 (R = (CH2)4NHBoc) was
prepared according to previous results by reduction of Fmoc
hydrazone, derived from the reaction of Fmoc carbazate with
aldehyde,31,37–39 while N-substituted Fmoc hydrazine 1 (R0 =
CH2CO2t-Bu) was obtained via nucleophilic substitution of
t-butyl bromo acetate by Fmoc carbazate.40 Then nucleophilic sub-
stitution of allyl bromo acetate 2 with Fmoc hydrazine 1 in the
presence of K2CO3 and NaI led respectively after four days at
80 °C to Fmoc-aza-b3-Lys(Boc)-OAllyl 3 (R = (CH2)4NHBoc) in 60%
yield or to Fmoc-aza-b3-Asp(t-Bu)-OAllyl 3 (R = CH2CO2t-Bu) in
⇑
Corresponding author. Tel.: +33 (0)23236933; fax: +33 (0)23236738.
E-mail address: michele.baudy-floch@univ-rennes1.fr (M. Baudy-Floc’h).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.11.113

776
S. Abbour, M. Baudy-Floc’h / Tetrahedron Letters 54 (2013) 775–778
TFA / DCM
K2CO3, NaI
R
N
R'
N
O
O
O
2
H
N
+
Fmoc
Fmoc
Fmoc
Br
1 / 1
68-87 %
N
H
R
Toluene
80°C
N
H
O
O
N
H
O
O
1
3
4
35-60 %
H2N
O
HO
O
O
4
Fmoc
N
Fmoc
N
N
H
O
N
H
4 R' = (CH2)4NH2
4 R' = CH2CO2H
Scheme 1. Synthesis of Fmoc-aza-b3-Lys-OAllyl and Fmoc-aza-b3-Asp-OAllyl.
O
O
O
DIEA
μW
O
HO
Fmoc
H
N
+
Br
Fmoc
O
N
N
H
OH
N
H
O
5
2
19 %
4 R' = CH2CO2H
Scheme 2. Synthesis of Fmoc-aza-b3-Asp-OAllyl by
lwaves activation.
O
Peptide chain assembly
O
H2N
O
Cl
using Fmoc/t-Bu chemistry
68%
4
N
H
Fmoc
Fmoc
N
N
4
N
H
O
N
H
DIEA
64%
4 R' = (CH2)4NH2
HN
O
H2N
O
H
N
NH
OH
O
O
TrtN
O
N
O
O
N
OH
O
NH
N
HN
O
O
O
a, b, c, d
55%
H
H
N
H
N
H
N
N
N
N
N
4
O
Fmoc
N
H
N
H
N
N
H
N
O
HN
H
3
4
NH
NH
O
BocHN
N
NBoc
O
PbfN
NHPbf
HN NH
NH2
C6W
NH2
Scheme 3. Synthesis of C6W: (a) Pd(Ph3P)4/PhSiH3 in DCM, 30 min twice; (b) 20% piperidine in DMF; (c) PyBOP/HOBt/DIEA in DMF; (d) TFA/TIS/H20.
35% yield.41 Selective deprotection with trifluoro acetic acid (TFA)
Trityl Chloride resin (CTC) (1 mmol, 1.54 mmol/g) was swollen in
DCM for 30 min and washed several times with dry DCM. Fmoc-
aza-b3-aa-OAllyl (1.2 equiv) was dissolved in dry DCM (10 mL)
and transferred to the resin bed. Following the addition of DIPEA
(4 equiv) via syringe, the mixture was gently agitated for 4 h at
room temperature. After draining, the resin was washed several
times with DMF and then with DCM. A capping step was proceeded
by addition of a mixture of DCM/MeOH/DIPEA (17/2/1). Then the
resin was washed with DCM (2 ꢀ 10 mL) and dried overnight in a
desiccator at room temperature. The substitution level was deter-
mined by UV spectroscopy.44
To demonstrate the feasibility of the on-resin cyclization of aza-
b3-peptides, we carried out the synthesis of a de novo cyclohexa-
pseudopeptide C6W (Scheme 3) and a cyclic aza-b3RGD analog
(Scheme 4). The side-chain immobilized Fmoc-aa-(CTC resin)-OAl-
lyl prepared above were used in the Fmoc/tBu-based synthesis. The
primary sequence was assembled using a microwave synthesizer
of Fmoc-aza-b3-Lys(Boc)-OAllyl 3 (R = (CH2)4NHBoc) or Fmoc-aza-
b3-Asp(t-Bu)-OAllyl
3 (R = CH2CO2t-Bu) afforded respectively
Fmoc-aza-b3-Lys-OAllyl 4 (R0 = (CH2)4NH2) in 68% yield or Fmoc-
aza-b3-Asp-OAllyl 4 (R0 = CH2CO2H) in 87% yield (Scheme 2).42
As the nucleophilic substitution proceeds in somewhat lower
yield (35% for Fmoc-aza-b3-Asp(t-Bu)-OAllyl 3 R = CH2CO2t-Bu),
we tried another way to get Fmoc-aza-b3-Asp-OAllyl
4
(R0 = CH2CO2H). Starting from Fmoc-aza-b3-Gly-OH 5 already de-
scribed,31 we realized a nucleophilic substitution of allyl bromo
acetate 2 in the presence of DIPEA. Even if the reaction was realized
on microwave activation (15 W, 2 h), the reaction proceeded in
19% yield, such a low yield of nucleophilic substitution by Fmoc
carbazate has already been observed previously. The Fmoc protect-
ing group must deactivate this reaction as we did not observe such
low yield with Boc carbazate.37
Fmoc-aza-b3-Lys-OAllyl, and Fmoc- aza-b3-Asp-OAllyl could be
loaded with a substitution level of 0.99 mmol/g and 1.05 mmol/g
respectively. The yield of 64–68% was due to the non activation
of the CTC resin before loading.43 Commercially available Chloro
utilizing
a single coupling, TBTU/DIEA activation protocol, a
four-fold excess of each amino acid residue, and conventional pro-
tecting groups. The removal of the allyl protection was carried out

S. Abbour, M. Baudy-Floc’h / Tetrahedron Letters 54 (2013) 775–778
777
O
O
Peptide chain assembly
O
Cl
using Fmoc/t-Bu chemistry
O
HO
Fmoc
O
Fmoc
N
N
N
H
N
H
O
DIEA
70 %
75 %
4 R' = CH2CO2H
O
O
O
a, b, c, d
66 %
O
O
c[Phe-Val-aza-β3Arg-Gly-aza-β3Asp]
Aza-β3 RGD
H
N
H
N
H
N
N
Fmoc
N
N
N
H
H
O
O
3
NH
NHPbf
PbfN
Scheme 4. Synthesis of aza-b3-RGD: (a) Pd(Ph3P)4/PhSiH3 in DCM, 30 min twice; (b) 20% piperidine in DMF; (c) PyBOP/HOBt/DIEA in DMF; (d) TFA/TIS/H2O.
according to the method by Albericio and co-workers.22 Two
30 min treatments at room temperature with a catalytic amount
14. Kuisle, O.; Quinoa, E.; Riguera, R. Tetrahedron Lett. 1999, 40, 1203–1206.
15. Kunz, H. Angew. Chem., Int. Ed. 1987, 26, 294–308.
16. Guibé, F. Tetrahedron 1998, 54, 2967–3042.
of Pd(PPh3)4 (0.1 equiv) and an excess of allyl acceptor phenylsi-
lane (24 equiv) in DCM resulted in clean removal of the allyl pro-
tecting group. To control the allyl deprotection a small amount of
resin was cleaved to afford the linear pseudopeptide N-Fmoc pro-
tected and the mass was checked by MALDI-TOF MS. The resin-
bound pseudopeptide, after the N-ter Fmoc deprotection, was used
in the on-resin cyclization with 4 equiv of PyBOP/HOBt and
10 equiv of DIEA in DMF overnight at room temperature (Schemes
2 and 3). The cyclic product was then deprotected and cleaved
from the resin support using a mixture of TFA/TIS/H2O (95/2.5/
2.5, v/v) for 3 h at room temperature. After precipitation by addi-
tion of cold ether and filtration, the final product of each reaction
was purified by rp-HPLC and its sequence as well as its purity
(>95%) was checked by MALDI-TOF MS for C6W (calcd [M+H]+:
912.528; found [M+H]+: 912.752) and for the aza-b3-RGD by HRMS
electrospray (calcd [M+H]+: 605.3160; found [M+H]+: 605.3160
(0 ppm)). Based on the initial loading, an overall yield of the on-re-
sin synthesis was calculated respectively at 23.9% and 34.7%.
In summary, we have demonstrated that Fmoc-aza-b3Lys-OAl-
lyl and Fmoc-aza-b3Asp-OAllyl could be easily prepared and conve-
niently used as a tether point for solid-phase linkage. With the
synthesis of two cyclo-aza-b3-peptides C6W and aza-b3-RGD we
demonstrated the feasibility of our synthetic approach that in-
cludes resin attachment of the first aza-b3-amino acid via its side
chain, successful use of combination of three orthogonal remov-
able protecting groups, stepwise Fmoc solid-phase synthesis of
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35. Laurencin, M.; Legrand, B.; Duval, E.; Henry, J.; Baudy-Floc’h, M.; Zatylny-
Gaudin, C.; Bondon, A. J. Med. Chem. 2012, 55, 2025–2034.
36. Neveu, C.; Lefranc, B.; Tasseau, O.; Do Régo, J.-C.; Bourmaud, A.; Chan, P.;
Bauchat, P.; Le Marec, O.; Chuquet, J.; Guilhaudis, L.; Boutin, J.; Ségalas-Milazzo,
I.; Costentin, J.; Vaudry, H.; Baudy-Floc’h, M.; Vaudry, D.; Leprince, J. J. Med.
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a
linear precursor aza-b3-peptide, and on-resin head-to-tail
cyclization.
References and notes
1. Gante, J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1699–1720.
2. Lee, H. S.; Syud, F. A.; Wang, X.; Gellman, S. H. J. Am. Chem. Soc. 2001, 123,
7721–7722.
3. Glatti, A.; Daura, X.; Seebach, D.; Van-Gunsteren, W. F. J. Am. Chem. Soc. 2002,
124, 12972–12978.
40. Busnel, O.; Bi, L.; Baudy-Floc’h, M. Tetrahedron Lett. 2005, 46, 7073–7075.
41. General procedure for the synthesis of Fmoc-aza-b3-aa-OAllyl 3: In a round-
bottom flask containing hydrazine 1 or 5 (1 equiv) in toluene (100 mL) were
added K2CO3 (0.8 equiv), NaI (2 equiv) and the allyl bromoacetate 2 (2 equiv).
The mixture was stirred 4 days at 80 °C. The solvent was then removed under
vacuum. Water and ethyl acetate were added to the mixture, and the organic
layer was washed with water and brine, dried on Na2SO4, and evaporated to
afford the crude product. Purification by CombiFlash chromatography
4. Kirshenbaum, K.; Zukermann, R. N.; Dill, K. A. Curr. Opin. Struct. Biol. 1999, 9,
530–535.
5. Liskamp, R. M. J. Angew. Chem., Int. Ed. 1994, 33, 633–636.
6. Semetey, V.; Rognan, D.; Hemmerlin, C.; Graff, R.; Briand, J. P.; Marraud, M.;
Guichard, G. Angew. Chem., Int. Ed. 2002, 41, 1893–1895.
7. Cheguillaume, A.; Salaun, A.; Sinbandhit, S.; Potel, M.; Gall, P.; Baudy-Floc’h, M.;
Le Grel, P. J. Org. Chem. 2001, 66, 4923–4929.
8. Laurencin, M.; Zatylny, Gaudin, C.; Henry, J.; Baudy-Floc’h. M. Fr. Demande
CNRS WO 2009/083663, 17 Avril 2009.
9. Baudy-Floc’h, M.; Le Blay-Laliberte, G.; Laurencin, M.; Deniel, F.; Barbier, G.
CNRS n 12/55899, 22 Juin 2012.
10. Laurencin, M.; Mosbah, A.; Fleury, Y.; Baudy-Floc’h, M. J. Med. Chem. in press.
11. Davies, J. S. J. Pept. Sci. 2003, 9, 471–501.
12. Li, P.; Roller, P. P. Curr. Top. Med. Chem. 2002, 2, 325–341.
13. Kuisle, O.; Quinoa, E.; Riguera, R. J. Org. Chem. 1999, 64, 8063–8075.
(Petroleum ether/ethyl acetate 55/45) led to
a
colorless oil. Fmoc-aza-b3-
Lys(Boc)-OAllyl 3 R = (CH2)4NHBoc (60% yield). 1H NMR (300 MHz, CDCl3) d
ppm: 1.33 (s, 9H), 1.40–1.42 (m, 4H), 2.81 (m, 2H), 3.01 (m, 2H), 3.64 (s, 2H),
4.12 (t, J = 6.5 Hz,1H), 4.36 (d, J = 6.5 Hz, 2H), 4.54 (d, J = 7.0 Hz, 2H), 4.69 (br,
1H), 5.17–5.28 (m, 2H), 5.76–5.89 (m, 1H), 6.78 (br, 1H), 7.18–7.33 (m, 4H),
7.50 (d, J = 6.7 Hz, 2H), 7.67 (d, J = 7.4 Hz, 2H); 13C NMR (75 MHz, CDCl3) d ppm:
170.4, 156.1, 155.9, 143.8, 141.3, 131.5, 127.7, 127.0, 125.1, 120.0, 119.1, 78.9,
66.7, 65.4, 57.5, 56.1, 47.2, 40.2, 28.4, 27.3, 24.6. HRMS (ESI) calcd for
C
29H37N3O6Na [M+Na]+: 546.25746; found 546.2575 (0 ppm). Fmoc-aza-b3-
Asp(t-Bu)-OAllyl 3 R = CH2CO2t-Bu (35% yield): 1H NMR (300 MHz, CDCl3) d

778
S. Abbour, M. Baudy-Floc’h / Tetrahedron Letters 54 (2013) 775–778
ppm: 1.38 (s, 9H), 3.62 (s, 2H), 3.76 (s, 2H), 4.13 (t, J = 7.1 Hz, 1H), 4.31 (d,
7.18–7.32 (m, 4H), 7.45 (d, J = 7.2 Hz, 2H,), 7.62 (d, J = 7.3 Hz, 2H), 10.03 (br,
2H); 13C NMR (75 MHz, CDCl3) d ppm: 170.0, 157.0, 143.4r, 141.3, 131.2, 127.8,
127.1, 124.9, 120.0, 119.1, 67.1, 65.8, 57.2, 56.2, 47.1, 39.9, 26.2, 24.2. HRMS
(ESI) calcd for C24H30N3O4 [M+H]+: 424.22308; found, 424.2230 (0 ppm). Fmoc-
aza-b3-Asp-OAllyl 4 R = CH2CO2H: (87% yield) 1H NMR (300 MHz, CDCl3) d ppm:
3.66–3.76 (m, 4H), 4.13 (t, J = 7.1 Hz, 1H), 4.34 (d, J = 7.1 Hz, 2H,), 4.53 (d,
J = 5.9 Hz, 2H), 5.16–5.30 (m, 2H), 5.76–5.90 (m, 1H), 7.13 (br, 1H), 7.20–7.36
(m, 4H), 7.47 (d, J = 7.3 Hz, 2H), 7.68 (d, J = 7.4 Hz, 2H). 13C NMR (75 MHz,
CDCl3) d ppm: 171.1, 169.9, 158.5, 143.2, 141.4, 131.0, 127.9, 127.2, 124.9,
120.1, 119.7, 67.9, 66.2, 59.7, 58.0, 47.0. HRMS (EI+) calcd for C22H23N2O6
[M+H]+: 411.15506; found, 411.1551 (0 ppm).
J = 7.1 Hz, 2H), 4.53 (dt, J = 5.8 Hz, 2H), 5.12–5.25 (m, 2H), 5.74–5.88 (m, 1H),
7.06 (br, 1H), 7.17–7.31 (m, 4H), 7.49 (m, 2H), 7.65 (d, J = 7.4 Hz, 2H).13C NMR
(75 MHz, CDCl3) d ppm: 169.3, 169.0, 155.3, 143.8, 141.3, 131.6, 127.7, 127.1,
125.2, 120.0, 118.9, 82.2, 67.1, 65.7, 57.9, 57.3, 47.1, 28.1. HRMS (ESI) calcd for
C
26H31N2O6Na [M+Na]+: 489.19961; found 489.1996 (0 ppm).
42. General procedure for the synthesis of Fmoc-aza-b3-aa-OAllyl 4: Monomer 3
(2 mmol) was dissolved in TFA/DCM (10 mL/10 mL) and the reaction was
stirred at room temperature during 5 h. The solvent was removed under
vacuum, and the crude product was purified by CombiFlash (CH2Cl2/MeOH:
90/10) to afford an orange oil. Fmoc-aza-b3-Lys-OAllyl 4 R = (CH2)4NH2 (68%
yield). 1H NMR (300 MHz, CDCl3) d ppm: 1.41 (m, 2H), 1.71 (m, 2H), 2.80 (m,
2H), 2.92 (m, 2H), 3.61 (s, 2H), 4.07 (t, J = 6.3 Hz, 1H), 4.33 (d, J = 6.3 Hz, 2H),
4.50 (d, J = 5.6 Hz, 2H), 5.13–5.23 (m, 2H), 5.72–5.82 (m, 1H), 6.99 (br, 1H),
43. García-Martína, F.; Bayó-Puxana, N.; Cruza, L. J.; Bohlingb, J. C.; Albericio, F.
QSAR Comb. Sci. 2007, 26, 1027–1035.
44. Hancock, W. S.; Battersby, J. E. Anal. Biochem. 1976, 1, 260–264.